G0-PCC-FISH derived multi-parametric biodosimetry methodology for accidental high dose and partial body exposures

High dose radiation exposures are rare. However, medical management of such incidents is crucial due to mortality and tissue injury risks. Rapid radiation biodosimetry of high dose accidental exposures is highly challenging, considering that they usually involve non uniform fields leading to partial body exposures. The gold standard, dicentric assay and other conventional methods have limited application in such scenarios. As an alternative, we propose Premature Chromosome Condensation combined with Fluorescent In-situ Hybridization (G0-PCC-FISH) as a promising tool for partial body exposure biodosimetry. In the present study, partial body exposures were simulated ex-vivo by mixing of uniformly exposed blood with unexposed blood in varying proportions. After G0-PCC-FISH, Dolphin’s approach with background correction was used to provide partial body exposure dose estimates and these were compared with those obtained from conventional dicentric assay and G0-PCC-Fragment assay (conventional G0-PCC). Dispersion analysis of aberrations from partial body exposures was carried out and compared with that of whole-body exposures. The latter was inferred from a multi-donor, wide dose range calibration curve, a-priori established for whole-body exposures. With the dispersion analysis, novel multi-parametric methodology for discerning the partial body exposure from whole body exposure and accurate dose estimation has been formulated and elucidated with the help of an example. Dose and proportion dependent reduction in sensitivity and dose estimation accuracy was observed for Dicentric assay, but not in the two PCC methods. G0-PCC-FISH was found to be most accurate for the dose estimation. G0-PCC-FISH has potential to overcome the shortcomings of current available methods and can provide rapid, accurate dose estimation of partial body and high dose accidental exposures. Biological dose estimation can be useful to predict progression of disease manifestation and can help in pre-planning of appropriate & timely medical intervention.

the cells were washed with RPMI-1640 and incubated for 2 h in PCC media (RPMI-1640 with 10% FBS, 1 μg/ml Colcemid).This was followed by a 6 min hypotonic treatment with KCl and fixation in 3:1 methanol-acetic acid.Detailed protocol for the same is reported previously 28 .Chromosomal spreads were prepared on a microscopic glass slide.Whole chromosome paint FISH was performed on freshly prepared slides as per manufacturer's protocol provided along with the probes.Chromosome pairs 1, 2 & 4 were painted in red, green and yellow (red + green) respectively using fluorescent DNA probes.Imaging was carried out with Axio-imager microscope Z-2, using automated metaphase capture software Metafer-5 from Metasystems (Germany) utilizing DAPI, FITC and SpO excitation emission filters for FISH.For analysis, IKAROS and ISIS platforms of Metasystems were used.
Number of fluorescent signals were counted among spreads with distinct FISH signal for each chromosome pair.Each signal was confirmed with individual fluorescence signal and counterstain to exclude false positives.Fluorescent spots, excess to two, were counted as damage or excess signal which could either be generated from breaks or mis-joining of breaks (translocations).In total, from three donors, either ≥ 100 aberrations or ≥ 500 cells were counted for each dose point.Frequency of excess signals was plotted against dose to generate the calibration curve.For validation of the generated calibration curve, scoring of five dose blinded samples was carried out using G 0 -PCC-FISH and dicentric assay.For G 0 -PCC-FISH, aberrations in 50-100 cells were counted and dose estimations were carried out with the equation obtained from the generated calibration curve.
Dicentric assay was performed in the following manner.Irradiated blood samples were allowed to repair for 2 h at 37 °C in humidified, CO 2 incubator.Subsequently, 0.5 ml blood was mixed with 4.5 ml RPMI medium supplemented with 10% FBS, and 10 μg/ml PHA.For metaphase arrest, long Colcemid treatment (0.02 μg/ml) was given, starting at 24 h till the end of the culture at 52 h.Metaphase harvesting was carried out as described previously (IAEA 2011).Either ≥ 500 cells or ≥ 100 dicentrics were counted in metaphase chromosomes for each sample.G2/M spreads without distinct centromere constrictions, where dicentrics were not clearly quantifiable, were ignored, irrespective of presence or absence of damage.

Simulated partial body exposure studies
Towards assessment of G 0 -PCC for its potential application in partial body dosimetry, studies were conducted on ex-vivo irradiated and simulated PBEs.Blood samples were collected from three donors as described in previous section, irradiated to 4 Gy, 8 Gy and 12 Gy of Co-60 gamma radiation.The simulation of PBE was carried out by mixing of exposed blood with unexposed blood.Variable proportions as well as variable doses were studied to assess both dose and proportion dependent effect on sensitivity and accuracy of the methods.At a fixed dose of 8 Gy, the proportion of unexposed fraction to the exposed fraction was gradually increased from, 1:0 to 1:1 to 1:3 and finally 1:5.Additionally, doses were varied from 4 to 8 Gy and 12 Gy at a fixed proportion of 1:1.Dicentric and G 0 -PCC-FISH assays were performed as described in previous section and G 0 -PCC-Fragment assay was performed as reported earlier 27 .Either ≥ 500 cells or ≥ 100 dicentrics were counted in metaphase chromosomes for each sample.For G 0 -PCC-FISH, 50-100 spreads were counted and for G 0 -PCC-Fragment, on an average, 50 spreads were counted.Aberration frequency, distribution of aberrations, and proportions of un-aberrated and aberrated cells were extensively analyzed and compared with the corresponding uniform exposure.The detailed methodology is presented in Fig. 1.In total, 54 simulated samples; 6 sets, from each of the 3 donors using the 3 methods, were analyzed.With the obtained data, G 0 -PCC-FISH was evaluated for its sensitivity for PBE, accuracy in dose estimation and its ability to discern a PBE.

Partial body dose estimation methodology
For dicentric assay, dose estimation was carried out using standard Dolphin's method 4,34 .CABAS tool was used for DCA based dose estimation, wherever possible.Accordingly, yield of aberration, Y, among the exposed fraction is given by: where; N is the total number of cells scored, X is the sum of all observed aberrations and n 0 is the total number of un-aberrated cells.
Extending the Dolphin's method to G 0 -PCC assay, the expected yield was calculated by introducing the correction for background level of aberrated cells (n bkg ).The Dolphin's method assumes that all the aberrated cells and aberrations come from the exposed fraction.This assumption is valid for dicentrics, as background level of aberrated cells are rare.However, in G 0 -PCC methods, background correction is included.
Hence, Eq. ( 1) is modified for G 0 -PCC as follows: The Yield, Y, obtained using Eqs.(1) and ( 2) is used for estimating the dose (D), as discussed in the following sections.
Response of aberrations in each donor and each of the chromosomes is also found to be linear quadratic, with negligible inter-individual variations.Overall aberrations combined for all doses, were highest for chromosome 1, followed by that for chromosomes 2 & 4 respectively.The frequencies of aberrations in chromosomes 1 & 2 were not statistically significant, although, the response of chromosome 4 was significantly lower than the other two chromosomes at multiple dose points, particularly beyond 2 Gy.Chromosome dependent responses are largely attributed to the genome size of the chromosomes.Similar results were reported earlier for metaphase based translocation assay 35 .Linear quadratic response for breaks and interchanges has been reported for chromosomes 3 and 4 using G 0 -PCC-FISH 29 , wherein the response was generated 8 h after irradiation to doses 1, 3, 5 & 7 Gy of gamma radiation.Another study has reported linear quadratic response in human chromosome no.8 using G 0 -PCC-FISH in chromosome 32 .A study limited to doses 2, 4 & 6 Gy for Chromosomes 1, 2 & 4 reported similar radiation responses 33 .In constrast to our data, one study has reported linear response for chromosomes 1 & 2 in the dose range 0-5 Gy using chemically induced G 0 -PCC-FISH 36 .In this study, relatively higher aberration yield for chromosome 2 than for chromosome 1 was also reported.The differential sensitivity between two chromosomes was not consistent in our data.The disagreement in the observation could be attributed to (1) difference in the degree of chromosome condensation between chemically vs. cell fusion induced G 0 -PCC, (2) scoring of limited number of cells from single individual and (3) limited dose range.
In G 0 -PCC-FISH, any number and combinations of chromosomes can be painted with chromosome specific color probes.The most popular ones are; one color, two color, three-color and Multi-plex FISH, which paint one, two, three chromosomes and entire genome (24 colors with 22 autosomes, X and Y) respectively.In this study, three color FISH has been carried out.Two, major considerations for the choice are; processing duration of FISH and sensitivity of the assay.Three color FISH requires same duration as one-or two-color FISH (< 24 h), and has more sensitivity (aberration/spread) due to higher genome proportion coverage (~ 22% compared to ≤ 8% and ≤ 16% in one-and two-color FISH respectively).With its higher sensitivity, it requires lesser number of spreads to be scored for dose estimation.While Multiplex FISH can be more sensitive needing only small number of spreads to be scored (20-25 for a uniform exposure), it demands longer duration (~ 48 h) for FISH processing, thus defeating the purpose of rapid biodosimetry.It is also likely that, for a localized exposure, counting < 50-100 cells may result in false negative dose estimation or incorrect dose, thereby not serving the purpose of scoring lesser number of cells either.Hence, three-color FISH appeared as a balanced option.It is worth emphasizing here that this is the first study on G 0 -PCC-FISH with multi-donor, broad range exhaustive calibration curve, enabling a robust statistical distribution of aberrations and accurate estimates of curve coefficients.Further, extensive dispersion analysis of aberrations from this data were performed which helped in identifying parameters to discern PBE from WBE.These results are discussed in a later section.

Validation of calibration curve
For validation of the calibration curve, dose estimations of five double blinded samples were carried out and the estimated doses were compared with those obtained from conventional dicentric assay (Table 1).Yield of aberrations (Y) was calculated from both the methods and dose was estimated for G 0 -PCC-FISH using Eq. 3, whereas for dose estimation with dicentrics, published curve equation (Eq.4) 37 was used: The G 0 -PCC-FISH method was found to be as accurate as dicentric assay in the tested range of 1-8 Gy, with all the estimates lying within 20% of the true value.Error in the dose estimates reduced with increasing dose.At lower doses, aberrations are relatively lower and contribution by background and individual response variations can result in higher errors compared to that at higher doses.

Simulated partial body exposure studies
Sensitivity of G 0 -PCC-FISH for partial body exposure Sensitivity of a method for PBE refers to efficiency to detect cells from the exposed fraction from the mixed pool.While un-aberrated cells may come from both exposed as well as un-exposed fraction, aberrated cells majorly represent the exposed fraction.Hence to evaluate the sensitivity of the three methods viz., dicentric assay, G 0 -PCC-Fragment and G 0 -PCC-FISH assay, we estimated proportion of aberrated cells in case of partial exposure (50%, 4 Gy, 8 Gy and 12 Gy) and plotted with that of corresponding uniform WBE of the same dose (Fig. 3).Table 1.Comparison of true dose and the doses estimated using conventional dicentric assay and G 0 -PCC-FISH assay.Values in the bracket represent estimates of lower limit (LL) and upper limit (UL) at 95% confidence and Error represents % deviation from the true dose.www.nature.com/scientificreports/It was observed that in case of dicentrics, sensitivity to detect aberrated cells from the exposed fraction was lower relative to the two G 0 -PCC methods.For instance, 4 Gy (WBE) sample has shown 60% ± 5.2 cells having dicentrics whereas in 50% exposed sample, only ~ 15% cells are found to have dicentrics instead of the expected ~ 30% (Fig. 3a).Efficiency has further declined with increasing doses; in case of 8 Gy and 12 Gy uniformly exposed samples, 100% cells are found to be aberrated whereas 50% exposed samples showed only < 10% and < 5% spreads having dicentrics.Further, in case of both G 0 -PCC methods, the proportion of cells with aberrations were in accordance with the proportion of exposed cells within ± 10% error (Fig. 3b, c).The lower sensitivity of dicentric assay can be explained on the basis of dose dependent decrease in cell division rate of exposed fraction and apoptosis in interphase, leading to a reduction in metaphase index (Fig. 3d).Hence, in such cases, while using conventional methods specialized equipment with automated high throughput imaging and scoring may be required to analyze huge number of cells 6,[38][39][40] .However, owing to higher sensitivity, G 0 -PCC has the potential to overcome this shortcoming of conventional assay with scoring of 50-100 spreads, which, can be easily scored in a simple, manual fluorescent microscope as well.

Dose estimations
T O evaluate accuracy of this method in partial body dosimetry, dose estimations from dicentric, G 0 -PCC-FISH and G 0 -PCC-Fragment assays were carried out and error in dose estimates were also calculated.For dose estimation, yields of aberration in the exposed fraction were calculated as described in the methodology section using Eqs.(1) and ( 2) and were subsequently used for calculation of corresponding dose estimates using appropriate curve equations.For G 0 -PCC-FISH and dicentric assay Eqs. ( 3) and ( 4) were used, whereas, for G 0 -PCC-Fragments the following equation, from a previously reported calibration curve 27 was used: Obtained estimates for each donor, with each method are plotted in Fig. 4a.In case of dicentric assay, severe underestimation (> 30% error) was observed at 12 Gy and 8 Gy, 1:5 proportion.Nevertheless, for 0, 1:1 & 1:3 proportion, dose estimates were close to true dose with scoring of ≤ 500 cells.Hence, it can be concluded that with increasing dose and decreasing exposed fraction, sensitivity of dicentric assay decreases and accuracy of dose estimate gets compromised.
Dose estimates in both G 0 -PCC-Fragments and G 0 -PCC-FISH did not suffer dose or proportion dependent underestimation.G 0 -PCC-FISH is found to be more accurate than G 0 -PCC-Fragment based dose estimation and background correction was needed to reduce the error at 1:5 proportion.In case of G 0 -PCC-FISH, all the dose estimates, except one, were within ± 20% error, with or without background correction.Further, with G 0 -PCC methods, due to high sensitivity, substantially low cell counts are sufficient.Hence, it can be inferred that G 0 -PCC methods are more suitable for high dose, PBE dosimetry, with the added advantage of being rapid.Specifically, G 0 -PCC-FISH estimated the doses with higher accuracy compared to G 0 -PCC-Fragment based technique.It is worth mentioning that while the calibration curves for G 0 -PCC-Fragments and G 0 -PCC-FISH are established up to 15 Gy, Dicentric assay is only up to 6 Gy.This raises a doubt whether the calibration curve could have contributed in underestimation of the doses in dicentric assay.The experimental results indicate that calibration curve holds good till 8 Gy although metaphase index may be lower; for 8 Gy dose point, as the proportion of unexposed fraction increased, accuracy of dose estimation was compromised, nevertheless, accurate estimates were obtained for uniform and 1:1 proportion.As anticipated, at 12 Gy, for uniform exposures, ~ 20% underestimation was observed while for PBE cases it was > 30%.In such a case, underestimation can be partly attributed to the calibration curve and partly to lower metaphase index from the exposed fraction.
Furthermore, in the methodology, we used background correction for both the PCC methods (Eq. 2) with the assumption that aberrated cell proportion also contains background level of aberrated cells from the unexposed fraction.When, unexposed fraction is large, the contribution of these cells resulted in considerable underestimation of doses at higher unexposed fractions, especially in G 0 -PCC-Fragment assay.
For G 0 -PCC fragment background level of aberration is ~ 20 per 100 cells (Eq.5), corresponding to 1 aberration for every 4 un-aberrated cells (n 0 ).Similarly, for G 0 -PCC-FISH considering the background level of aberration of 0.057 cells (c in Eq. 3), 6 aberrations per 100 n 0 were estimated as background.
Accordingly, for every background count, one single aberrated cell (n 1 ) was considered as background (n bkg ) and added to the cells with n 0 .The associated aberrations were also subtracted from total aberrations.Cells with more than 1 aberrations were counted as background when n 1 were inadequate.It is noteworthy that, for low dose exposures the un-aberrated cells are contributed from exposed fraction as well.Hence, background correction should be done only after ascertaining PBE of doses ≥ 4 Gy.
To demonstrate utility of the background correction, % errors in dose estimates were plotted with or without background correction as shown in Fig. 4b.Background correction was more impactful in keeping the errors within 30% in G 0 -PCC-Fragment method, especially where un-exposed fractions were higher (8 Gy, 1:5) as observed in Fig. 4b(i).This can be attributed to higher background aberration frequency in unexposed fraction in G 0 -PCC-fragment compared to that in G 0 -PCC-FISH.G 0 -PCC-FISH practically did not benefit from background correction in our study, where the probability of contribution of background is very low at all proportions (Fig. 4b(ii)).Error remained within 20% for all the dose estimates except one in both corrected as well as un-corrected estimates.However, the equation has been maintained with background correction so it remains independent of the exposed fraction and can be used for both the PCC methods.
It is noteworthy that all G0-PCC assays have been conducted 24 h after exposure with scientific and practical reasons.Previously it was reported that optimal duration between exposure and processing or collection of samples is about 8-24 h based on chromosomal break repair kinetics 28 .In addition, for a PBE, lymphocyte pool recirculation time also needs to be considered.One circulation of peripheral blood takes only < 1 min to travel from and back to the heart.Hence, for a localized exposure, lasting for more than couple of minutes, immediately collected blood will not represent true PBE.However, only about 2% of lymphocytes are present in the peripheral blood while the remaining are present in lymphoid pool, which is relatively stagnant.Equilibration of lymphocytes between these two pools requires a duration of about 8-12 h, and the blood can be collected following this period.Furthermore, considering the delays attributed to detection of incident, reporting, logistic responses and transporting the exposed individual or reaching the site for collection of samples, it is also a most practical time window wherein the sample can be collected by biodosimetry team or medical professionals.

Dose estimation for PBE based on the discerning key parameters
Differentiation of PBE from WBE is very crucial for dose estimation.For this, we compared the dispersion of aberrations among WBE and PBE of the same dose and their deviations from Poisson were quantified by www.nature.com/scientificreports/Papworth's U-test (Fig. 5a, b, c).On applying Papworth's U-test, it was observed that, in case of WBE, though overdispersion was observed, the distribution was closer to Poisson whereas, in all the PBE samples, considerable deviation from Poisson was observed (U-value range: 5.4-32.9).Deviations in WBE were moderate and hence the Dolphin's method was able to provide dose estimates within reasonable uncertainty.U-test has been considered to be a good indicator of PBE in case of dicentrics.However, as observed in Fig. 5, signals counted in G 0 -PCC may not strictly follow Poisson distribution and large overdispersions may be observed at lower doses.As a result, U-value can only be indicative of PBE.Therefore, instead of exclusive reliance on U-test, a multiparametric approach should be considered when using G 0 -PCC for discerning PBE.
After comparing the distribution of aberrations among WBE and PBE, we could identify three key parameters that can be used in combination to differentiate the two types of exposures for a given yield of aberrations; (i) median of aberrations among aberrated cells (P1), (ii) proportion of multi-aberrant cells (with ≥ 3 aberrations) among aberrated cells (P2) and (iii) proportion of un-aberrated cells among total scored cells (P3).The first two are dependent more on the dose to the exposed fraction and remain largely un-affected by the amount of exposed fraction.The three parameters show a specific response under uniform exposure which is plotted in Fig. 6.For a given whole body dose estimate (D WBE ), substantial deviation from the expected response of key parameters can confirm non-uniformity.Figure 6 shows that P1 was 1 for doses ≤ 2 Gy, and further increased in a linear quadratic manner.On the other hand, P2 follows a sigmoidal pattern wherein, up to 2 Gy, majority of the aberrations are contributed by single aberrated cells; while beyond 2 Gy, multi-aberrant cells increase exponentially up to 8 Gy before saturating to ~ 100%.In contrast, P3 is > 90% for doses below 1 Gy, and decreases exponentially with increasing dose.
In Fig. 7, we have demonstrated that different WBE and PBE samples, with different exposure scenarios, can result in similar yield leading to similar D WBE ; nevertheless they can still be differentiated based on the key parameteres.For example, D WBE of 2 ± 0.1 Gy is possible under any one of the following three scenarios, (i) 2 Gy WBE, (ii) 4 Gy 1:1 PBE and (iii) 8 Gy 1:5 PBE.For this example, the total cell population and aberrant cell population, along with the key parameters are plotted in Fig. 7a.As may be seen from the aberrant cell population, the P1is 1, 2 and 4 for 2 Gy and 4 Gy 1:1 and 8 Gy 1:5 respectively while the corresponding P2 values are 10%, 33% and 63%.The dose estimates corresponding to these two paramters (see Fig. 6) are ≤ 2 Gy, 3.3-4.0Gy and 6.8-7.0Gy respectively.On the other hand, the data from total cell population shows unexpectedly high value of P3 which is also indicative of PBE.Similarly, in Fig. 7b, 6 Gy WBE and 12 Gy 1:1 PBE yields give rise to similar dose estimates but difference in the three key parameters again confirms the non-uniformity of exposure in the latter case.Hence, with the above example, it can be concluded that key parameters have potential to distinguish a partial exposure from a uniform exposure.It is noteworthy that besides discerning the nature of exposure, median and % multi-aberrant cells can also be used for dose estimation.Once, PBE is confirmed, dataset can be corrected for background for further dose estimation with Dolphin's approach.Also, P1 and P2 from the corrected dataset can provide more accurate dose estimation.
A step by step methology for discerning the type of exposure and dose estimation of an unknown sample is disscused in the next section.
Stepwise protocol for partial body dosimetry of an unknown sample post G 0 -PCC-FISH: an example Sample preparation: Blood sample with unknown dose is collected.PBMCs are isolated and fusion with mitotic cells is performed.2 h post fusion, cells are harvested, treated with hypotonic, and fixative.Slides are prepared and FISH is performed.Excess fluorescence signals are counted.For details of G 0 -PCC-FISH refer to methodology section and 28 .
Example dataset: Assume that the scoring of aberration results in the following dataset: Where n 0 , n 1 … refer to cells with 0, 1 … aberrations, X refers to total number of aberrations and N is total number of cells.
Initial dose estimation: Yield, Y is calculated as X/N.Assuming WBE and the dose estimate (D WBE ) of is obtained by solving Eq. (3).D WBE = 2 Gy.Decision on WBE/PBE: All the key parameters expected from 2 Gy WBE were deduced from Fig. 6.Observed values of key parameters were extracted from the scored data.The doses corresponding to the observed values were estimated from standard curves in Fig. 6.Disagreement between observed and expected values was an indication of PBE as presented below:

PBE dose estimation:
Once PBE is confirmed, dose estimation is performed with background correction.Background aberrations were calculated as 6% of n 0 hence, 11 single aberrated cells from n 1 population were considered as background (n bkg ).These cells are added with n 0 cells and their aberrations subtracted from total aberrations (corrected X = 182, N − (n 0 + n bkg ) = 35).Further, corrected P1 and P2 were calculated to be 5 and 78.3% respectively.Yield of the exposed fraction was calculated from Dolphin's approach by solving Eq. 2 & dose estimates were obtained using Eq. 3, P1 and P2 as shown below: (1) Dolphin's approach: D = 8.4 Gy   is partial body in nature.The gold standard biodosimetry method, dicentric assay has limited applicability at high doses.It tends to underestimate the dose with increasing extent of non-uniformity as well as dose to the exposed fraction.Moreover, it requires 48-52 h of culture, needs large number of cells to be scored and hence, may effectively need 3-5 days for dose estimation.In the present study, it is demonstrated that G 0 -PCC offers higher efficacy and sensitivity to detect PBE at high doses.In comparison, 500-> 1000 metaphases need to be analyzed in dicentric assay, 50-100 PCC spreads were found to be sufficient for dose estimation at all the simulated nonuniform exposures.The limitation of dose and non-uniformity dependent underestimations are not observed for G 0 -PCC in the tested range.G 0 -PCC based chromosomal aberration analysis is not subjected to cell cycle arrest and no cell culture setup is required, hence, dose estimates can be obtained in less than 24 h.G 0 -PCC-FISH is found to be more accurate than G 0 -PCC-Fragment.Three key parameters were identified which can discern a PBE and can also provide a close dose estimate.In conclusion, G 0 -PCC-FISH is rapid, sensitive method which can distinguish a PBE from uniform WBE and provide multi-parametric accurate dose estimation.Limitations of the method include the need of mitotic cells and optimized cell fusion protocols.The study has been carried out with homogeneous gamma radiation to the exposed part however, accidental conditions can be complex including High LET exposures, mixed radiation type of exposures or protracted exposures for which limitations and advantages of this method need to be explored.Further, dose estimations of in-vivo samples should be carried out for further validation of the methodology.
https://doi.org/10.1038/s41598-024-65330-8www.nature.com/scientificreports/dependent increase in chromosomal aberrations was distinctly visible.Representative images of G 0 -PCC-FISH spreads with increasing frequency of aberrations are presented in Fig.2a.The calibration curve from pooled data of the three donors is plotted in Fig.2bwhile the individual data of the three donors is plotted in Fig.2c.Further, radiation response for each of the chromosomes 1, 2 & 4 for each of the donors is plotted in Fig.2d.The calibration curve follows a linear quadratic response, with the Yield, Y, being given as:

( 4 )Figure 2 .
Figure 2. Ex-vivo radiation response of chromosome 1, 2 & 4 associated breaks & interchanges in human PBMCs assessed using G 0 -PCC-FISH.(a) Microscopic images of PCC spreads showing increasing number of aberrations with increasing dose (left to right).(b) Calibration curve from pooled data of aberrations of three donors.(c) Individual response curves of three donors and d) Individual response curves of the three chromosomes, pooled from three donors.The error bar represents mean ± Poisson Error.

Figure 3 .
Figure 3. Sensitivity of different methods for detection of aberrant cells from the exposed fraction in PBE relative to WBE of the same dose in in-vitro simulated exposures using human blood lymphocytes.The proportion 1:1 refers to samples with 50% exposed proportion.(a) dicentric assay (b) G 0 -PCC-Fragments (c) G 0 -PCC-FISH.(d) Typical 10 X fields of metaphase preparation in dicentric assay with increasing uniform exposure.Metaphases are encircled in red boundaries.For both the PCC methods, data was corrected for background level of aberrated cells in case of PBE.Data bars represent mean ± SD. https://doi.org/10.1038/s41598-024-65330-8

Figure 4 .
Figure 4. Dose estimates among simulated PBE samples (a) Dose estimates of three different donors for all the three methods.1:0, 1:1, 1:3, 1:5 refers to ratio of exposed vs. unexposed fraction of the blood.Small and bigger orange ellipticals refer to 20% and 30% error vertically.Doses were heavily underestimated in case of dicentric assay at 12 Gy, 1:1 in all the three donors and at 8 Gy, 1:5 in two of the three donors.(b) Impact of background correction in dose estimation using G 0 -PCC-methods.(i) G 0 -PCC-FISH (ii) G 0 -PCC-Fragments.While there is no significant impact of background correction in G 0 -PCC-FISH, dose estimation error is significantly reduced in G 0 -PCC-Fragments, specially at higher unexposed proportions.

Figure 5 .
Figure 5. Distribution of aberrations for a given dose with WBE and after 1:1 mixing with unexposed cells (50% PBE).(a) 4 Gy, (b) 8 Gy, (c) 12 Gy andd) Quantification of dispersion with U-test among three donors.U-value within ± 1.96 refers to Poisson Distribution (dark green cells) greater value refers to greater deviation from Poisson (red cells).

Figure 6 .
Figure 6.Radiation response of the key parameters under WBE condition: (i) P1, Median of aberrations among aberrated cells shows a linear quadratic response (ii) P2, proportion of multi-aberrant cells (X ≥ 3) among aberrated cells shows a sigmoidal response and (iii) P3, proportion of un-aberrated cells among total cell population depicts an exponential decrease.